A yeast TCP-1-like protein is required for actin function in vivo.

Abstract

We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits ofcytosolc heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-tye Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enh the phenotypes of actin mutants. Taken together, these findin demonstrate that TCP-l-like proteins are required for actin and tubulin function in vivo.TCP-1 (t-complex polypeptide 1) is a subunit of eukaryotic cytosolic chaperoning. It has sequence homology to the subunits of bacterial (GroEL/hsp60), plastid, and mitochondrial (chaperonin 60) chaperonins (1, 2), protein complexes that facilitate protein folding in cells (3-5). The structure of the cytosolic chaperonin, a two-layered torus composed of multiple subunits (6)(7)(8), is similar to that of the bacterial, mitochondrial, and plastid chaperoning, but the subunit composition is more complex. Recent studies have identified distinct families of TCP-1-related proteins, referred to as CCT (chaperonin containing TCP-1) proteins (9). Within a family, the CCT proteins share -60%o sequence identity across species lines, while between families the identity is only =30%6, even within a single species such as mouse or yeast.TCP-1-containing chaperonins from rabbit, mouse, and bovine tissues can facilitate the folding of newly synthesized actin and tubulin (8, 10, 11) as well as actin-related and tubulin-related proteins (12). In addition, in CHO cell lysates, tubulin and a 43-kDa protein that comigrates with actin on polyacrylamide gels are bound to chaperonins (13 (20) to create pDV211. A nested deletion series was created from pDV211 by using Exo III and was used for sequencing the sense strand ofANC2 (nt 1-2727; Biomolecular Resource Center sequencing facility, University of California, San Francisco). The antisense strand of ANC2 was sequenced from nt 2307-439 by using oligonucleotide primers.ANC2 Deletion and Genetic Linkage Analysis. The ANC2 null allele was generated by replacing nt 530-2305 (EcoNI/ Hpa I fragment), which include the entire chromosomal ANC2 coding sequence plus 194 bp upstream, in two different wild-type diploid strains (DDY426 and DDY665) with the integrating plasmid pDV219, which contains the LEU2 gene with ANC2 flanking sequences at the 5' (BamHI/EcoNI; 530 bp) and the 3' (Hpa I/BamHI; 435 bp) ends. A total of 60 tetrads derived from six independent transformants for both parental strains ...

Functional Interaction between Phosducin-like Protein 2 and Cytosolic Chaperonin Is Essential for Cytoskeletal Protein Function and Cell Cycle Progression

“…Figure 3A shows that plp2-ts alleles exhibit sensitivity to both latrunculin and benomyl, indicating microfilament and microtubule defects, respectively. Similar phenotypes are also observable in strains carrying ts or cold-sensitive (cs) alleles of several CCT genes (our unpublished observations; Ursic et al, 1994;Vinh and Drubin, 1994).…”

“…Although CCT impacts a variety of substrate proteins, its most abundant and best characterized substrates are the cytoskeletal proteins actin and tubulin, and, accordingly, yeast ts alleles of CCT display various actin-and tubulinrelated cytoskeletal defects (Gao et al, 1992;Ursic et al, 1994;Vinh and Drubin, 1994;Siegers et al, 1999;Spiess et al, 2004). We therefore predicted that if Plp2p modulates the function of CCT, by way of direct physical interaction (Figure 1, A and B), the plp2-ts alleles might also exhibit cytoskeletal anomalies.…”

Hsp60C is required in follicle as well as germline cells during oogenesis in Drosophila melanogaster

“…The most significant abnormalities observed in Hsp60C 1 mutant or mo- (Chen et al, 1994;Melki and Cowan, 1994;Vinh and Drubin, 1994;Liang and MacRae, 1997;Sarkar et al, 2006). Furthermore, Hsp60 family proteins, immunoprecipitated with the same anti-Hsp60 antibody as used by us, were found to bind most strongly with actin in C. elegans (Leroux and Candido, 1997).…”

The Tetrahymena chaperonin subunit CCTη gene is coexpressed with CCTγ gene during cilia biogenesis and cell sexual reproduction

“…In vivo studies indicate that newly synthesized a-and 13-tubulin and actin enter in a 900 kDa complex containing TCP1 and that tubulin is released from this complex competent to form heterodimers [21]. In yeast, S. cerevisiae, the mutations in CCT~, CCT8 and CCT~( cause abnormal microtubular structures [22][23][24] and disruption of actin microfilaments [23].…”